The present invention relates to membrane polypeptides, methods of preparation, and assays that employ them.
Cell membranes are made of lipids capable of forming a barrier between aqueous compartments. They consist primarily of a continuous double or bilayer plate of lipid molecules associated with various membrane proteins. The phospholipids, sphingolipids, and glycolipids make up the three major classes of membrane forming lipid molecules. These lipids are amphipathic (amphiphilic) molecules in that they have a hydrophilic (polar) head and a hydrophobic (non-polar) tail. In the aqueous environment of cells, the polar head groups face toward the water while their hydrophobic tail groups interact with each other to create a lamellar bilayer, and to a lessor extent other aggregate structures depending on the lipid composition and conditions. For example, membrane lipids can form a variety of different shapes including spheres (vesicles), rods (tubes) and lamellae (plates) depending on lipid and water content, and temperature. These shapes represent basic units that interact to form two- and three-dimensional lattice matrix structures classified as lamellar phase (e.g., bilayer plate, closed sphere), hexagonal phase (e.g., rod), or cubic phase (e.g., spheres, rods or lamellae connected by aqueous channels) (Lindblom, et al., Biochimica et Biophysica Acta (1989) 988:221-256). A cross section of a typical cell membrane bilayer (lamellar phase) of phospholipid can be viewed as having a hydrophobic core region of about 30 Angstroms (xc3x85) with two interfacial regions of about 15 xc3x85 each (White, et al., Curr. Struc. Biol. (1994) 4:79-86).
Proteins can associate with cell membranes in different ways. Integral membrane proteins contain at least one component that is embedded within the lipid bilayer. The non-polar segments of these integral membrane proteins, which embed in the lipid bilayer perpendicular to the surface of the membrane, may consist of a hydrophobic region of the polypeptide, a covalently attached fatty acid chain or other types of lipid chains. Peripheral membrane proteins normally associate with the lipid bilayer through non-covalent interactions with these integral membrane proteins. Additionally, some peripheral membrane proteins are located entirely in the aqueous phase, associated with the membrane through a covalently attached fatty acid or lipid chain. The co-translational attachment of a fatty acid chain such as myristic acid to the amino-terminal glycine of a protein through an amide linkage results in localization of the protein to the cytoplasmic face of cellular membranes. Prenyl groups and palmitic acid groups are attached post-translationally via thioether linkages to cysteine residues and also result in localization of proteins to the membrane. These types of covalent attachments are important for function in a wide variety of cell signaling proteins, like the heterotrimetric G proteins (James, et al., Biochemistry (1990) 29(11):2623-2634; Morello, et al., Biochem. Cell Biol. (1996) 74(4):449-457; Mumby, S. M., Curr. Opin. Cell. Biol. (1997) 9(2):148-154; Resh, M. D., Cell Signal (1996) 8(6):403-412; and Boutin, J. A., Cell Signal (1997) 9(1):15-35). Glycosylphosphatidylinositol anchors, found at the C-terminus of soluble proteins, result in the attachment of these proteins to the cell surface membrane (Turner, A. J., Essays Biochem. (1994) 28:113-127).
The two major classes of known integral membrane proteins are those that insert xcex1-helices into the lipid bilayer, and those proteins that form pores in the lipid bilayer by xcex2-barrel strands (Montal, et al., Curr. Opin. Stuc. Biol. (1996) 6:499-510; Grigorieff, et al., J. Mol. Biol. (1996) 259:393-42; and Weiss, et al., J. Mol. Biol. (1992) 227:493-509). Single membrane spanning proteins, or single-pass membrane proteins, generally have a hydrophobic region that anchors that sequence in the lipid bilayer via an xcex1-helix configuration. Multiple membrane spanning proteins, or multi-pass membrane proteins, result from the polypeptide chain passing back and forth across the lipid bilayer and typically employ xcex1-helix and/or xcex2-barrel structured membrane anchors.
Examples of membrane proteins include membrane-associated receptors, transporter proteins, enzymes, and immunogens. For instance, cell membrane-associated receptors represent a dynamic collection of membrane proteins of particular therapeutic importance. Four basic superfamilies are recognized: the enzyme-linked receptors, the fibronectin-like receptors, the seven transmembrane receptors, and the ion channel receptors. Enzyme-linked receptors represent single-pass membrane proteins, with the basic structure consisting of a single polypeptide traversing the plasma lamella once via an xcex1-helix anchor domain. The extracellular domain of enzyme-linked receptors binds hormone/ligand, while the carboxyl-terminal domain contains a catalytic site that promotes signal transduction via hormone/ligand binding and receptor aggregation.
The fibronectin-like receptors have the same general structure as the enzyme-linked receptors except that no specific catalytic site is represented in the cytoplasmic domain. Class 1 fibronectin-like receptors contain two modified extracellular domains formed from two seven-stranded xcex2-sheets that join at right angles to create a ligand-binding pocket. The class 2 fibronectin-like receptors have a slightly different structure in that they form repeats of five-stranded xcex2-sheets that extend over the hormone like fingers. The class 1 and 2 receptors contain a conserved proline-rich cytosolic juxtamembrane region that constituatively binds soluble tyrosine kinases, which is activated by ligand/hormone-binding and receptor aggregation.
The seven-transmembrane receptors, also called G-protein coupled receptors, serpentine receptors, or heptahelical receptors, represent the largest and most diverse family of membrane receptors identified to date. These receptors mediate sensory and endocrine related signal transduction pathways and are multi-pass membrane proteins having xcex1-helical anchor regions that transverse the membrane seven times. The transmembrane spanning regions for some of these proteins form a small ligand/hormone-binding pocket, while larger binding sites are formed through extended amino terminal regions. Seven-transmembrane receptors also contain one or more intracellular loops that bind and activate G-proteins, which act as second messengers in cells.
The ion channel receptors are represented by the ligand- and voltage-gated channel membrane protein receptors. Ligand-gated ion channels are formed by pentamers of homologous subunits. Each subunit contributes an xcex1-helix toward forming the wall of the channel. Ligand/hormone binding appears to occur between the subunits. The typical voltage-gated channel receptors are homotetramers, with each subunit having six transmembrane xcex1-helices.
Different techniques have been used to study membrane proteins and/or exploit them for therapeutic purposes, diagnostics, and drug screening assays and the like. However, unlike non-membrane proteins, the biggest obstacle in working with membrane proteins is the poor solubility of their hydrophobic polypeptide chains, the difficulty in folding membrane proteins from unfolded polypeptide chains and the difficulty in overexpressing and isolating them in environment suitable for quantitative analyses (Huang, et al., J. Biol. Chem. (1981) 256:3802-3809; and Liao, et al., J. Biol. Chem. (1983) 258:9949-9955). For example, unfolding and folding whole transmembrane proteins is difficult since they are insoluble in the lipid bilayer in the unfolded form, as well as in the aqueous phase in both their folded and unfolded forms, because of their highly hydrophobic character (Haltia, et al., Biochimica et Biophysica Acta (1995) 1241:295-322). This feature of membrane proteins is particularly problematic when attempting to synthesize, label or otherwise manipulate them chemically in a cell free environment. Nevertheless, individual transmembrane segments of membrane proteins have been chemically synthesized via solid phase chemistry, followed by subsequent insertion into membranes and spontaneous assembly of native-like structures with biological activity (Popot, et al., Biochemistry (1990) 29:4031-4037; and Grove, et al., Methods Enzymol. (1992) 207:510-525). To date, however, solid phase synthesis has been limited to synthesis of only a few short transmembrane peptide segments, since membrane proteins are recalcitrant to standard chemical synthesis techniques.
Establishing access to membrane proteins with site-specific chemical modifications is crucial both for the analysis of structure-function relationships of membrane proteins and for drug discovery. The most important techniques currently employed to achieve this goal are the synthesis of small membrane-spanning peptide fragments of these proteins (Grove, et al., Methods Enzymology (1992) 207:510-525; and MacKenzie, et al., Science (1997) 276:131-133), chemical modification of existing or engineered cysteine residues (Oh, et al., Science (1996) 273:810-812), and in vitro suppression mutagenesis to incorporate unnatural amino acids (Cload, et al., Chemistry and Biology (1996) 3:1033-1038; and Turcatti, et al., J. Biol Chem. (1996) 271:19991-19998). None of these techniques provides general access to totally synthetic or semi-synthetic membrane proteins containing chemically modified amino acid side-chains, or their production in a quantity sufficient for most biophysical techniques. Additionally, such techniques do not permit modular synthesis and reassembly of membrane-incorporated transmembrane polypeptide segments or domains.
Wilken, et al. (Curr. Opin. Biotech. (1998) 9(4):412-426) review chemical protein synthesis. Dawson, et al. (Science (1994) 266:776-779) disclose chemical synthesis of water-soluble polypeptides by native chemical ligation. Grove, et al. (Methods in Enzymology (1992) 207:510-525) disclose Boc-chemistry solid phase synthesis of small pore forming membrane peptides and their subsequent incorporation and activity in a lipid membrane. MacKenzie, et al. (Science (1997) 276:131-133) disclose recombinant synthesis and radioactive labeling of the transmembrane domain of glycophorin A and its incorporation and NMR structure in a lipid membrane. Oh, et al. (Science (1996) 273:810-812) disclose NMR structure of a diptheria toxin transmembrane domain by chemical modification of existing or engineered cysteine residues with a methanethiosulfate spin label to generate a nitroxide side chain. Turcatti, et al. (J. Bio. Chem. (1996) 271:19991-19998) disclose in vitro suppression mutagenesis in Xenopus oocytes to introduce fluorescence-labeled amino acids into the seven transmembrane neurokinin-2 receptor and its incorporation and activity in oocyte membranes. Portman, et al. (J. Phy. Chem. (1991) 95:8437-8440) disclose incorporation and activity of xcex1-chymotrypsin and bacteriodopsin in a cubic phase lipid matrix. Giorgione, et al. (Biochemistry (1998) 37(8):2384-2392) disclose incorporation and activity of protein kinase C in a cubic lipidic phase matrix and liposome.
The present invention relates to methods and compositions for lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides, compositions produced by the methods, and assays that employ them. The methods involve contacting a lipid matrix-incorporated membrane polypeptide with a ligation label comprising one or more amino acids, where the polypeptide and label comprise amino acids having unprotected reactive groups capable of chemoselective chemical ligation. A variety of chemoselective chemistries can be used for ligation such as native chemical ligation, oxime-forming ligation, thioester forming ligation, thioether forming ligation, hydrazone forming ligation, thiazolidine forming ligation, and oxazolidine forming ligation. Compositions of the invention include totally synthetic and semi-synthetic lipid matrix-embedded membrane polypeptides that are produced by the lipid matrix-assisted chemical ligation method of the invention.
The present invention further includes a method of forming a lipid matrix-embedded membrane polypeptide comprising a ligation site amenable to chemoselective chemical ligation when treated with a reagent that cleaves the polypeptide directly adjacent to a residue amenable to chemoselective ligation. This aspect of the invention involves contacting a membrane polypeptide that is embedded in a lipid matrix with a reagent that selectively cleaves the polypeptide at a specific site so as to generate a lipid matrix-embedded membrane polypeptide with an unprotected N-terminal or C-terminal residue that is amenable to chemoselective chemical ligation. The cleavage site may occur naturally in the polypeptide or the polypeptide can be engineered to contain one or more such sites.
The present invention also includes a method of detecting a ligand that directly or indirectly interacts with a folded membrane polypeptide embedded in a lipid matrix. This aspect of the invention involves contacting with a ligand, a lipid matrix-embedded synthetic or semi-synthetic membrane polypeptide produced by lipid matrix-assisted chemical ligation, where the ligand and/or the membrane polypeptide comprise a detectable label. The ligands may be derived from any number of sources including naturally occurring ligands and synthetic and semi-synthetic sources, such compound libraries. This method is particularly useful for diagnostic assays, screening new compounds for drug development, and other structural and functional assays that employ binding of a ligand to a prefolded membrane polypeptide.
Also provided is support matrix suitable for screening assays, where the support matrix comprises a detectably labeled lipid matrix-embedded membrane polypeptide attached thereto through a chemical handle. The present invention also provides kits having at least one or more compositions of the invention.
The present invention further provides a method for on-resin labeling a peptide with a chelator-sensitized metal ion probe. The method involves labeling one or more amino acids of a peptide attached to a resin with a zwitterionic chelator moiety label capable of chelating metal ions. Also included is a method to increase the solubility of a zwitterionic chelating agent. This method involves combining an insoluble zwitterionic chelating agent with a solubilizing agent that produces a soluble salt form of the zwitterionic chelating agent. The invention also provides s a composition comprising a soluble salt form of a zwitterionic chelator agent.
The methods and compositions of the invention permit unprecedented access to membrane polypeptides and their site-specific labeling with one or more detectable labels. The methods and compositions also have multiple additional uses. For example, they can be used to assay ligand binding to membrane polypeptides and domains comprising a receptor, and thus are extremely useful for structure/function studies, drug screening/selection/design, and diagnostics and the like, including high-throughput applications. The methods and compositions of the invention are particularly suited for FRET analyses of previously inaccessible membrane polypeptides.